Anti nkp46

Immune cells of 3-month-old male mice were isolated from spleen, kidney, bone marrow and thymus by flushing and pelleting (bone marrow) or by mechanical dissociation followed by centrifugation through a percoll gradient (kidney, spleen and thymus). Erythrocytes were lysed with ammonium-chloride-potassium lysing buffer, and the resulting leukocytes were counted on a haemocytometer. Cells were stained for flow cytometry analysis with anti-CD45 (Becton Dickinson, ref: 25-0451-82, dilution: 1/1,000), anti-B220 (eBioscience, ref: 45-0452-80, dilution: 1/1,000), anti-CD11b (eBioscience, ref: 12-0112-82, dilution: 1/1,000), anti-CD11c (eBioscience, ref: 11-0114-85, dilution: 1/1,000), anti-NKp46 (eBioscience, ref: 48-3351-82, dilution: 1/1,000), anti-CD8 (eBioscience, ref: 17-0081-81, dilution: 1/1,000), anti-TCRβ (Becton Dickinson, ref: 563221, dilution: 1/1,000) and Fixable Viability Dye (eBioscience, ref: 65-0865-14). Samples were acquired on CyAnADP 9 flow cytometer (Beckman Coulter) using Summit acquisition software (V). Data were analysed using Kaluza flow analysis software V (Beckman Coulter). An example of this analysis is shown in Supplementary Fig. 12.

Anti-Nono (Cat# 11058-1-AP and 66361-1-Ig) was from Proteintech. Anti-Elk3 (Cat# NBP1-83960) was from Novus Biologicals. Anti-Tmem241 (Cat# 203644-T32) was from Sinobiological. Anti-Lineage cocktail (Cat# 88-7772-72), Anti-CD127 (A7R34), anti-Sca-1 (D7), anti-Flt3 (A2F10), anti-α₄β₇ (DATK32), anti-Id2 (ILCID2), anti-PLZF (Mags.21F7), anti-Eomes (Dan11mag), anti-NKp46 (29A1.4), anti-NK1.1 (PK136), anti-CD45 (30-F11), anti-CD25 (PC61.5), anti-Gata3 (TWAJ), anti-RORγt (AFKJS-9), anti-Bcl11b (1F8H9), anti-CD3 (17A2), anti-CD19 (1D3), anti-KLRG1 (2F1), anti-CD90 (HIS51), anti-IL-22 (IL22JOP), anti-Ki67 (SolA15), anti-CD45.2 (104), anti-BrdU (BU20A), anti-PD-1 (J43) and anti-CD45.1 (A20) were purchased from eBiosciences (San Diego, USA). Anti-c-Kit (2B8), anti-CD150 (TC15-12F12.2), anti-CD48 (HM48-1), and anti-CD49a (HMα1) were purchased from Biolegend (California, USA). Anti-β-actin (Cat# RM2001) was purchased from Beijing Ray Antibody Biotech. Paraformaldehyde (PFA) and 4’,6-diamidino-2-phenylindole (DAPI) were from Sigma. IL-22 ELISA kit was purchased from Neobioscience.

For flow cytometric analysis, CBA mice were i.p. infected with 2 × 10⁵ PFU of TCV MCMV. Splenocytes were extracted and prepared as previously described [73 (link)] and incubated with 2.4G2 monoclonal antibody to reduce nonspecific staining. The following monoclonal antibodies were used for cell surface staining: anti-CD3ε (eBioscience), anti-NKp46 (eBioscience), anti-CD11b (eBioscience), CD27 (BD), anti-CD69 (eBioscience).
To assess NK cell proliferation, CBA mice were i.p. injected with 2 mg of BrdU (Sigma) and splenocytes extracted 2 h later. To detect BrdU incorporation, prepared splenocytes were first stained for surface antigens prior to fixation, permeabilisation, refixation, treatment with DNase I and intracellular stained according to manufacturer’s instructions (BrdU flow kit; BD, Franklin Lakes, USA).
To detect IFNγ production by NK cells, splenocytes were incubated in RPMI supplemented with 10% fetal calf serum (FCS) for 5 h in the presence of 500 IU/mL of IL-2 and 1 μg/mL of brefeldin A (BFA; eBioscience) at 37 °C. Cells were surface stained, then fixed and permeabilised using Cytofix/Cytoperm solutions (BD Pharmingen, San Diego, CA, USA) followed by intracellular staining according to manufacturer’s instructions.